Cytomegalovirus is the leading infectious cause of birth defects which can result in deafness and mental retardation in neonates, and can cause severe viral pneumonia and colitis in transplant recipients and sight-threatening retinitis in patients with AIDS. Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with a number of cancers including Burkitt lymphoma, nasopharyngeal carcinoma, Hodgkin lymphoma, and post-transplant lymphoproliferative disease. Human CMV and EBV naturally infect humans, but not small animals or nonhuman primates. The best models currently available for CMV and EBV are rhesus monkey CMV and EBV. The goal of this study is to develop an effective vaccine for these rhesus viruses and to use these as a model for vaccines for their human counterparts. We are using various approaches including soluble recombinant proteins, recombinant virus vectors expressing viral proteins, and replication defective viruses as vaccines. We have been using an animal model, rhesus monkey EBV in rhesus macaques, to compare various candidate EBV vaccines. Rhesus EBV causes a similar disease in monkeys as EBV does in humans and the viruses have the same number of virus genes that have virtually the same activities. We have constructed three different rhesus EBV vaccines. First, we produced soluble rhesus EBV glycoprotein gp350 (gp350) which is the principal virus protein that is important for attachment of EBV to cells. Soluble rhesus EBV gp350 was chosen since this vaccine candidate is furthest along in clinical trials in humans. Second, we produced virus-like replicon particles (particles that can infect cells and produce proteins, but not spread from cell to cell) that express rhesus EBV gp350. This vaccine was predicted to induce strong antibody and T lymphocyte responses to gp350. Third, we produced VRPs that express rhesus EBV gp350, rhesus EBV EBNA-3A, and rhesus EBV EBNA-3B. These latter two proteins induce the highest level of T lymphocyte responses to EBV in healthy persons. Animals vaccinated with soluble rhesus EBV gp350 produced the highest titers of antibody to the glycoprotein. Animals vaccinated with VRPs expressing rhesus EBV EBNA-3A and EBNA-3B developed virus-specific T lymphocyte immunity to these proteins, while animals vaccinated with VRPs expressing rhesus gp350 did not have detectable T lymphocyte immunity to gp350. After challenge of the animals with wild-type rhesus EBV, animals vaccinated with soluble rhesus EBV gp350 showed the best level of protection followed by animals that received VRPs expressing rhesus EBV gp350, EBNA-3A, and EBNA-3B as assessed by seroconversion or levels of rhesus EBV DNA or RNA in the blood shortly after challenge. At 2 years after challenge, animals that received gp350 that became infected had lower levels of rhesus EBV DNA in the blood compared with those receiving the other vaccines. Since the level of EBV DNA in the blood is predictive for EBV lymphomas in transplant patients, the ability of gp350 to reduce the level of EBV in the blood of rhesus macaques after infection suggests that this vaccine might have a role in reducing certain EBV-associated cancers. Thus, the initial response to virus challenge may be predictive of the viral load years after vaccination. We have also developed a candidate vaccine virus for rhesus CMV in which we deleted a protein from the virus that is essential for virus growth. The resulting replication-defective virus lacks one viral protein, glycoprotein L (gL), and replicates only in cells expressing rhesus CMV gL. In addition, noncomplementing cells infected with the replication-defective rhesus CMV produced glycoprotein B, the major target of neutralizing antibodies, at levels similar to those observed in cells infected with wild-type virus. We plan to test this candidate vaccine in rhesus monkeys.